In the past, protein sequencing has been carried out by techniques such as those involving the sequential removal of amino acids from one end of the protein and identifying each removed amino acid in turn. Other techniques have relied on the genetic code, using the base sequence of the gene coding for the protein. Both these techniques are slow, complex and difficult.
More recent techniques have attempted to obtain amino acid sequence information using mass spectrometry, typically using fast atom bombardment to ionize the sample. In fast atom bombardment, a sample dissolved in a liquid is bombarded with atoms or ions. Charged molecules resulting from this process are directed into the spectrometer and detected. An example of this technique is described in the text entitled "Macro Molecular Sequencing and Synthesis Selected Methods and Applications", 1988, published by Alan R. Liss, Inc., specifically at pages 83 to 99 in an article in such text entitled "Mass Spectrometry in Bio-Pharmaceutical Research" by Steven A. Carr et al.
A difficulty with the technique using mass spectrometry is that when complex protein molecules are fragmented, analysis of the daughter or fragment ions has been extremely difficult. As noted by Carr et al at page 86 of the above identified test, a Y-B analysis technique can be used to determine sequence information. However the analysis is complex, slow and difficult, and so far as the applicant is aware has never been commercially used.
According to the invention an improved method of analyzing proteins is provided, utilizing ion evaporation followed by tandem mass spectrometry. The method of the invention provides tryptic fragments which are predominantly doubly charged, one charge being located at each end of the fragment. Such fragments are then further fragmented into two singly charged daughter fragments or daughter ions in the tandem mass spectrometer, providing information which can be much more readily used to obtain the sequence of the amino acids in the protein.